Polysaccharides are natural macromolecular compounds comprising of a plurality of monosaccharide molecules bonded via glycosidic bonds, and are one of the fundamental substance of life. In recent twenty years, with the development of molecular biology and cell biology, it was discovered that polysaccharides have a variety of biological functions, polysaccharides and their conjugates are involved in the life activities of cells, such as cell-specific recognition, component of a variety of antigens and drug receptors on cell surface, activation of immune cells. Thus polysaccharides arouse great research interests.
Polysaccharides of higher plants and fungi have a long history of applications in China, and have also very rich resources. They has become a focus and a research hotspot. Modern pharmacological studies indicate that these two types of polysaccharides have very important and special physiological activities, and play a clear role in promoting immunity, anti-bacterial, anti-viral, anti-parasitic, anti-tumor, anti-radiation, anti-thrombotic, anti-clotting, anti-aging, inflammation, lowing blood fat and improving animal fertility and other aspects. Moreover, most of the polysaccharides have no direct cytotoxicity, and can be used in long-term. Polysaccharides of higher plants and fungi have currently become one of the most promising health care resources.
Bioactivity of polysaccharides largely determines the application value of polysaccharides. Constituent components, composition and spatial conformation, molecular weight and molecular weight distribution, water-solubility of polysaccharides are the main factors affecting biological activities of polysaccharides. Numerous studies show that the molecular weight of active polysaccharides is one of the necessary conditions for having biological activity. Greater molecular weight of polysaccharide, greater apparent volume of the molecule, which does not benefit polysaccharides to go across multiple membrane barriers into play in vivo biological activities. Water-soluble polysaccharides closely related to the molecular weight thereof is another important condition for playing its biological activities.
The very complex structure of polysaccharides causes the synthesis thereof extremely difficult. Currently, all active polysaccharides are extracted from natural products. Higher plants polysaccharides and fungi polysaccharides are extracted and isolated from different plants or different parts of the same plant, and fungal sporocarp, mycelium and mycelial fermentation broth.
There is no commonly accepted, effective, unified separation method of extracting polysaccharides. Existing processes can generally be summarized as water extraction, acid extraction, alkali extraction, salt extraction and enzyme supplementary extraction. These methods have significant shortcomings in the aspects of polysaccharide extraction efficiency, cleanness of the production process, energy and material consumption, or polysaccharide structure modification, etc. The technology value of obtained polysaccharide products is not very high (particularly in: polysaccharide's low purity, poor water solubility, broad molecular weight distribution, etc.), which limits the high value-added applications of polysaccharides. Specifically, the existing polysaccharide extraction methods have the following problems:
Water extraction generally is time-consuming, has high energy consumption, uses a large amount of extracting solvent, and has low polysaccharide extraction yield.
It is hard to control the amount of inorganic strong acid, strong base and reaction time in acid alkali extraction, which easily causes the activity of polysaccharide molecules be destroyed, and even makes polysaccharides generate pigment molecules of small molecular weight burdening the subsequent bleaching work. Moreover, after the end of the reaction, neutralization or dialysis to acid, alkali solution much be quickly done, otherwise it will cause products contaminated, and increase insecurity of the products for food and healthcare products. In addition, the use of non-degradable inorganic acid or base likely causes serious environmental pollution in large scale industrial production.
The enzyme used in enzyme assisted extraction is generally expensive, often is easily inactivated, and has other shortcomings like short life and low purity. In enzymatic hydrolysis process, the optimal temperature is often in a very small range, and slight fluctuations of reaction conditions may cause the enzyme activity significantly decrease. Therefore, enzyme extraction has relatively high requirements on experimental conditions, and even requires very complicated pretreatment to extraction raw materials. Enzyme extraction technology still needs further research to be used for the industrial extraction of polysaccharides.
There are many reasons for these difficulties. In general, higher plants, fungi active polysaccharides have large molecular weight, and poor water-solubility. They have low content in the plant materials, complex distribution condition and distribution status, wherein some are in free state, some are bonded with macromolecules like proteins and hemicellulose to form complex conjugates, some are in the cytoplasm, some are in the cell wall. Main components of plant cell walls are cellulose, and other substances including hemicellulose, pectin, lignin, etc. Cellulose has supermolecular stable structure of high degree crystalline region, which is difficult to be hydrolyzed. Common extraction method can only apply a large number of solvent for long time immersion, so that the full expansion of the plant cell wall will make compact structure becomes loose and reduce the mass transfer resistance of the active ingredients diffusing from the cell to the solvent. Thus, conventional methods have high energy and material consumption, utilize polysaccharides in a free state in plants, but have poor extraction effects on polysaccharide wrapped in call wall or bonded in certain forms with other macromolecules.
CN03133778.3 patent application discloses a method of fully releasing active ingredients of lucid ganoderma spores. The method puts lucid ganoderma spore into a microwave reaction chamber, adds an organic acid solution, conducts microwave treatment after mixing, then vacuum distills to remove the organic acid, and finally uses conventional water extraction, alcohol precipitation method to extract crude lucid ganoderma spore polysaccharides from microwave treated lucid ganoderma spore. Compared with conventional extraction methods, the method exponentially increases the yield of polysaccharides (above 3 times), but has the following main problems. First, only using vacuum distillation rather than further washing with an organic solvent to remove residual acid after the microwave treatment causes the resulting product having a high content of residual acid. And in the removal of oxalate acid, calcium precipitation method is adopted, which first washes off oxalic acid from the spores with water along with dissolved polysaccharide; when calcium ions are added to form calcium oxalate precipitates, a small amount of polysaccharides would be wrapped and lose. Second, the mechanism is based on that microwave enhanced reaction between organic acid and chitin and glial in the cell wall of lucid ganoderma spore, as to reduce restrictions of these substances on polysaccharide release, but does not recognize the importance of breaking the chemical bonding between polysaccharides and protein, cellulose, hemicellulose, and chitin, and degradation of polysaccharides on improving the yield and water-solubility of polysaccharides.
U.S. Pat. No. 8,110,677 discloses a method of microwave extraction of active polysaccharides from artemisia songarica schrenk. The method has technical defects like using a large amount of extraction solvent (water of 30-50 times is needed), and that enzymolysis is needed after obtaining polysaccharides, wherein enzymolysis causes many constraints, such as long reaction time (10-12 hours in the patent), and removal of enzyme after the reaction (n-butanol and chloroform extraction in the patent).
CN200510026889.1 patent application discloses a method of microwave extraction of astragalus polysaccharides. The method uses an inorganic strong acid (hydrochloric acid or sulfuric acid), an inorganic strong base (potassium hydroxide, sodium hydroxide, ammonia), wherein the inorganic strong acid and inorganic strong base cause serious equipment corrosion, are difficult to be recycled, and easily cause environment pollution, and the amount of inorganic acid is still relatively large. The hydrolysis of polysaccharides by the inorganic acid or base is mainly performed by adjusting the acid concentration, they only have acids, alkaline degradation and bond cleaving effects on polysaccharides, and the hydrolysis of polysaccharides under inorganic strong acid and strong base conditions is difficult to control, and cannot achieve the protective effects on polysaccharide molecules.
Therefore, there is a need to further develop a new method to extract active polysaccharides from higher plants or fungi.